Non-serum culture medium for multiple animal cell large-scale culture

A serum-free medium, large-scale culture technology, applied in the direction of animal cells, tissue culture, microorganisms, etc., can solve the problems of low yield, high cost and high price in the production process

Inactive Publication Date: 2008-01-16
EAST CHINA UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] (1) Serum is susceptible to contamination by viruses, mycoplasma or other pathogens;
[0005] (2) Differences between different batches of serum cause differences between product batches;
[0006] (3) The existence of a large amount of serum protein increases the difficulty of downstream separation and purification, which increases the cost and reduces the recovery rate;
[0007] (4) It is difficult to completely remove some serum proteins by means of separation and purification, which seriously affects the final quality of the product
[0012] (1) Due to the high price, it is only suitable for small-scale use in laboratories; it is not suitable for large-scale bioreactors;
[0013] (2) The medium is not optimized for cells, and the supported cell density is low, resulting in low yield in the production process;
[0014] (3) Although some media can support cell growth well, it often leads to the reduction or even loss of the ability of cells to express products;
[0015] (4) The medium is highly specific to cells, and usually a medium is only suitable for one cell line or cell line, but not for other cell lines or cell lines

Method used

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  • Non-serum culture medium for multiple animal cell large-scale culture
  • Non-serum culture medium for multiple animal cell large-scale culture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The serum-free medium of the present invention can be used for high-density continuous perfusion culture of hybridoma cells, and the composition of the medium is as follows:

[0036] Element

Chinese chemical name

Inorganic salt (mg / L)

CaCl 2

calcium chloride

116.60

CuSO 4 ·5H 2 o

copper sulfate

0.0013

Fe(NO 3 ) 2 9H 2 o

Ferric nitrate

0.05

FeSO4 7H 2 o

ferrous sulfate

0.417

KCl

potassium chloride

311.80

MgCl 2

magnesium chloride

28.64

MgSO 4

magnesium sulfate

48.84

NaCl

Sodium chloride

6995.50

NaHCO 3

sodium bicarbonate

2440

NaH 2 PO 4 h 2 o

Sodium dihydrogen phosphate

62.50

Na 2 HPO 4

Disodium phosphate

71.02

ZnSO 4 ·7H 2 o

Zinc sulfate

0.432

L-amino acid (mg / L)

Alanine

Alanine

4.45

Arginmin...

Embodiment 2

[0042] The serum-free medium of the present invention can be used for high-density continuous perfusion culture of rCHO cells, and the composition of the medium is as follows:

[0043] Element

[0044] Na 2 HPO 4

[0045] (NH 4 ) 6 Mo 7 o 24 4H 2 o

[0046] rCHO cells (rCHO SS3 A2, expressing human anticoagulant factor m) were subcultured and adapted in the serum-free medium according to the present invention, and then inoculated in a 2-liter bioreactor of B.BRAUN at an inoculation density of 2.0×10 5 cells / ml, start perfusion after 40 hours of cultivation, the perfusion rate is 0.58 (1 / day), the perfusion medium is the serum-free medium of the present invention, and the cell density is maintained at 0.9-1.0×10 after 255 hours of cultivation 7 The cells / ml is about 350-380U / L, and the product concentration is maintained at about 350-380U / L (see Figure 2). Compared with the results of ordinary medium batch culture, the cell density an...

Embodiment 3

[0048] The serum-free medium of the present invention can be used for the cultivation of 293 cells, and the composition of the medium is as follows:

[0049] Element

[0050] Fe(NO 3 ) 3 9H 2 o

[0051] Thiarnine HCl

[0052] After the 293 cells were subcultured and adapted in the serum-free medium of the present invention, they were inoculated in a 2-liter bioreactor of B.BRAUN for batch culture, and the inoculation density was 2.45×10 5 cells / ml, it can be seen from Figure 3 that there is almost no lag phase in the growth of the cells, and they enter the exponential growth phase after inoculation, with an average specific growth rate of 0.46day -1 , with a maximum viable cell density of 11.0×10 5 cells / ml. In the figure, curve 7 is the living cell density, and curve 8 is the ratio of living cells.

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Abstract

The invention opened non-serum mediums which can proper to culture many kinds of the animal cells. The character of it is to use the DMEM / F12 as the base medium, then add the growth factor, hormone, the essential amino acid and the microelement. It has the functions: (1) it can support many cells and clones; (2) it is same as the serum medium in the growth of the cell and the expression of the product; (3) it support the long heritable culture; (4) it is benefit for the isolation of the product because it contains less protein; (5) it is cheap. We can get the high density of the cells and the product concentration using the medium.

Description

technical field [0001] The invention relates to a serum-free medium used for producing biological products such as antibodies, vaccines, and gene recombinant proteins during large-scale high-density culture of animal cells. technical background [0002] Animal cell culture has been widely used in the production of various biologically active substances such as monoclonal antibodies, virus vaccines, virus vectors, immune regulators, growth factors, specific tumor antigens, and various gene recombinant protein drugs. Add a certain amount (5%-10%) of bovine serum, which contains growth factors, hormones, carrier proteins, adhesion factors, trace elements and other nutrients required for cell growth, which can greatly promote cell growth and product expression. [0003] However, the application of serum also brings many disadvantages: [0004] (1) Serum is susceptible to contamination by viruses, mycoplasma or other pathogens; [0005] (2) Differences between different batche...

Claims

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Application Information

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Patent Type & AuthorityPatents(China)
IPC IPC(8): C12N5/06C12N5/07
Inventor谭文松朱明龙周燕华平牛红星
OwnerEAST CHINA UNIV OF SCI & TECH